DHX8 在 DNA 損傷反應中之角色

Abstract

DNA 損傷反應能夠去偵測到損傷，並且誘導一連串的信號去啟動 DNA 損傷檢查點，進而去進行修復，以維持基因體的穩定性，而其中細胞對 DNA 損傷的反應主要受到 ATR-Chk1 信號路徑調控。然而， DNA 損傷反應的失調可能導致修復不完全、突變的產生和加速衰老，從而影響人類遺傳性疾病的發展。 DHX8 是一種 RNA 解旋酶，參與在 mRNA 的剪接和調節成熟 RNA 從細胞核中的釋放。在我們的研究中， Dr. Wu 和我發現當細胞暴露在不同的基因遺傳毒性的物質時，DHX8 的缺失會導致 ATR-Chk1 信號路徑的激活產生缺陷。我還發現沉默 DHX8 基因並不會影響參與 DNA 切除的蛋白質表現量，且 RPA 複合物徵召至 ssDNA 的能力顯著下降。這可以解釋為什麼當 DHX8 基因沉默時 ，ATR-Chk1 信號路徑的激活是有缺陷的。此外，即使在去除 CPT 並且修復8小時， HeLa 細胞中 DHX8基因的沉默也顯示出持續性的 γH2AX foci 。總之，這部分結果表明，在基因毒性的壓力下， DHX8 對於激活 ATR-Chk1 信號路徑和 DNA 修復是很重要的。在我的第二部分的研究中，我發現 DHX8 會被 SUMO2/3 修飾。我使用定點突變 PCR 和 IP 進一步研究，顯示出 DHX8 在賴氨酸 140 和賴氨酸 399 處被 SUMO 2/3修飾。有趣的是，紫外線照射可以增強 DHX8 的 SUMO修飾。這表明DHX8的SUMO 2/3修飾可能在 DDR 中具有重要的作用。在本研究中，我利用定點突變的方法將 SUMO 2/3修飾所需的兩個賴氨酸殘基突變。我發現 DHX82KR 突變體無法挽救細胞的生長缺陷，這表明 DHX8 的 SUMOylation 修飾可能在細胞增殖或細胞存活中起作用，這部分還需要更多實驗來釐清。除了 SUMOylation 之外，本研究的質譜分析還發現 DHX8 在 UV 照射下，可以在絲氨酸 460 和蘇氨酸 554 處被磷酸化。無論 DHX8 的磷酸化是否在 DDR 或 DNA 損傷中起作用，DHX8對 RNA 代謝的調節都需要更多的研究來被闡明。

The DNA damage response (DDR) senses the damage and induces a series of signaling pathways to activate the DNA damage checkpoint and repair DNA damage to maintain genomic stability. Therefore, dysregulation of the DNA damage response could cause incomplete repair, generation of mutations, and accelerated aging, which can lead to the development of human genetic disorders. DHX8 is an RNA helicase involving in splicing of mRNA and regulation of the release of mature RNA from the nucleus. In this work, Dr. Wu and I found that the loss of DHX8 resulted in the defects of ATR-Chk1 activation when cells were exposed to different genotoxic agents. The level of proteins involved in DNA resection was not altered after DHX8 knockdown, while the recruitment of RPA proteins to the ssDNA was significantly abolished. This may explain why the activation of ATR-Chk1 pathway was defective when DHX8 was silenced. Moreover, silence of DHX8 in HeLa cells showed persistent γH2AX foci even at 8 hours after removal of CPT. Together, this part of work showed that DHX8 is important for activation of the ATR-Chk1 pathway and the DNA repair upon genotoxic stress. In my second part of work, I found that DHX8 is conjugated by SUMO2/3. Further investigation using site-directed mutagenesis PCR and IP, I confirmed that DHX8 is SUMOylated at lysine 140 and lysine 399 by SUMO2/3. Interestingly, SUMOylation of DHX8 can be enhanced by UV irradiation. This suggests that the conjugation of DHX8 by SUMO2/3 may play a role in the DDR. I further generated DHX8 mutants, in which two-lysine residues of the domain for SUMO modification were conserved to arginine. I found that this DHX82KR mutant cannot rescue the growth defect. It suggests that DHX8 modification by SUMO may have a role in the regulation of cell proliferation or cell survival. Moreover, mass spectrometry analysis also indicates that DHX8 can be phosphorylated at serine 460 and threonine 554 upon UV irradiation. The role of phosphorylation of DHX8 in the DDR or DNA damage requires further studies.